Exposure to environmental chemicals or chemotherapeutic agents can cause DNA damage that results in toxicity or carcinogenicity. While the underlying mechanisms are in some cases at least partially understood, in many other instances they remain unclear. The broad objective of this proposal is to understand how chemical injury to the genome leads to activation of cell death pathways. Of particular interest is how topoisomerase II (topo2) poisons, which cause double-stranded DNA breaks and are among the most effective and widely prescribed antineoplastic agents, lead to the engagement of the mitochondrial (intrinsic) apoptotic pathway. Further, additional information on the underlying mechanisms will be obtained by investigating how cells made resistant to these agents due to an overexpression of Bcl-2 escape cell death. It is well known that activation of the mitochondrial apoptotic pathway is accompanied by the release of cytochrome c and activation of caspase-9. Although it is believed that activation of caspase-9 within the Apaf-1 apoptosome represents the first or apical caspase event in this pathway, the candidate's recent findings demonstrating that caspase-2 activity occurs upstream of mitochondrial cytochrome c release and caspase-9 and -3 activation in response to the topo2 poison etoposide suggest that this may not be generally true. These findings have led to the hypothesis that caspase-2 activation is required for engagement of the mitochondrial apoptotic pathway in response to DNA-damaging agents, and this activation is regulated by Bcl-2. The specific aims are: 1) to determine whether caspase-2 is initially activated within the nucleus in response to topo2 inhibition and then translocates to the cytosol where it engages the mitochondrial apoptotic pathway; 2) to determine if caspase-2 is an initiator caspase and can induce apoptosis independently of the Apaf-1 apoptosome and/or caspase-3 activation; and 3) to determine if Bcl-2 overexpression prevents the appearance or accumulation of drug-induced DNA damage and thereby regulates caspase-2 activation and cell death. These studies will be carried out, in part, using short interfering RNA (siRNA) to down-regulate procasp-2 and apaf-1, flow cytometry to measure cell cycle progression and apoptotic markers, expression and purification of recombinant active and inactive caspase-2 proteins, and confocal microscopy to evaluate caspase-2 activation and redistribution. Completion of this research will provide critical new information about the specific molecular mechanisms whereby DNA-damaging agents activate cell death pathways.